Ongoing studies in this project concern the cell biology, biochemistry and molecular biology of Leishmania, a group of protozoan pathogens of humans. All Leishmania parasites undergo a dimorphic life cycle: 1) in mammals (humans), they multiply as obligate intracellular amastigote forms within the lysosomal system of macrophages, eventually destroying these cells and 2) within their insect vectors (blood-sucking sandflies), they differentiate and multiply as, extracellular promastigote forms within the alimentary tract and eventually migrate to the mouth parts for transmission. By World Health Organization estimates, Leishmania parasites annually cause well-over 12 million cases of human disease (leishmaniasis) worldwide. In infected humans, these parasites destroy macrophages within the skin or internal organs (i.e. spleen, liver and bone marrow) causing either large and disfiguring, malignant skin ulcers (e.g. caused by L. mexicana) or degenerative and most often fatal visceral disease (e.g. caused by L. donovani). Previous studies from our laboratory have established that Leishmania parasites constitutively secrete over 40 different soluble protein, glycoprotein and carbohydrate constituents. Such secretory products can readily permeate throughout and presumably alter the host micro-environments in which Leishmania reside. Thus, an understanding of the nature of these parasite products seems essential. To that end, parasite secretory enzymes are investigated toward defining their functional roles in the survival, maintenance, growth and transmission of these organisms. Further, genes encoding these proteins have been identified and characterized toward defining their expression and regulation during parasite growth, development and differentiation. In that context, previously we identified and characterized the genes encoding the unique Leishmania secretory chitinase enzyme family. In FY 2008-09, we used combined biochemical and molecular approaches to demonstrate that this enzyme family was conserved among all pathogenic species of Leishmania. This suggested that they must play significant functional roles in the growth, development and survival of all members of this group of human pathogens. In that regard, in continuing collaborative studies, in FY 2008-09, we examined the ability of L. mexicana chitinase over-expressor parasite mutants to survive and grow within a permissive sand fly vector host. Our observations showed that these chitinase over-expressor transfectants were:1) able to escape from the fly-host peritrophic membrane compartment at a faster rate;2) produce considerably higher parasite burdens in the sandfly gut;3) cause greater damage to the sand fly stomodeal valve and 4)to produce larger lesions in infected mice than control parasites. Cumulatively, these data showed that the parasite chitinase acts as a multifunctional virulence factor for L. mexicana by facilitating its survival and transmission in its sandfly vector. In other on going collaborative studies, attempts are being made to functionally delete the gene for the L. mexicana chitinase and to examine the viability and survivability of these mutants in both a mammalian (mouse) host and sandfly vectors. In addition, previously, we demonstrated that virtually all Leishmania sp., like other trypanosomatid parasites are purine auxotrophs and therefore are, totally dependent upon salvaging these essential compounds from their insect vector and mammalian hosts. Thus in FY 2008-09, we identified and characterized the biochemical and functional properties of a unique new, 35 kDa,secretory nuclease from L. mexicana. Our studies showed that this enzyme was constitutively released/secreted by both amastigotes and promastigote developmental forms of this parasite. Using a molecular approach, we identified, characterized the gene, LmexNucS that encodes this new Class I nuclease family member from these organisms. Sequence analysis revealed that LmexNucS possesses a signal peptide and five structural motifs characteristic of the P1/S1 fungal/plant secretory nuclease family. Northern blot and protein analyses confirmed that LmexNucS was transcribed and differentially translated through the parasites life cycle (Amastigotes>>Promastigotes). Western blot and enzyme activity analyses verified that LmexNucS was constitutively secreted/released by both L.mex Pro- and Amastigote developmental forms. In order to delineate the functional properties of the LmexNucS, the gene was episomally over-expressed in LmexNucS-HA transfectants. Results of combined anti-HA immunoprecipitation/ enzyme activity assays showed that LmexNucS was N-linked glycosylated and that it could readily degrade RNA, single stranded DNA, double stranded DNA as well as various synthetic polynucleotide substrates (i.e. poly-A, -I, and -U). Further we demonstrated that LmexNucS was irreversibly inactivated by sulfhydryl reducing agents e.g. DTT. Cumulatively, our results indicate that the L.mexNucs must play important roles in facilitating the growth, development and survival of this important human pathogen. In that regard, this leishmanial secretory nuclease might be exploited for diagnostic or for therapeutic purposes. In FY2008-09 we were also involved in a collaborative project concerning the proteomic analysis of the secretome of L. donovani. In these studies, quantitative mass spectrometry was used to analyze the proteins released/secreted by various developmental forms of L. donovani during their growth in vitro. Analyses resulted in the identification that more that 151 proteins that are apparently released/secreted by these parasites. In silico analyses showed that most of these exosomal proteins did not possess an amino-terminal signal peptide a motif typically associated with the classical eukaryotic ER-targeted secretory pathway. Cumulatively, results of this study indicated that protein secretion in Leishmania is a heterogeneous process, which is mediated by both classical signal peptide pathways as well as by multiple non-classical secretion pathway mechanisms including the release of exosome-like microvesicles. Taken together, the results of our recent and ongoing studies continue to provide pertinent and significant information toward understanding the unique pathophysiology of these parasites. In addition, these studies are of practical relevance toward demonstrating whether specific /unique parasite enzymes and regulatory proteins are logical targets for 1) the design of new chemotherapeutic drugs, 2) the development of new diagnostic tools and/or 3) useful as potential vaccines against these human pathogens.